Nitrile hydratases catalyze the addition of one molecule of water to the nitrile, resulting in the formation of the corresponding amide according to Reaction 1:R—CN+H2ORCONH2  Reaction 1
Similarly, methods for producing carboxylic acids are known and use microorganisms that produce an enzyme that possesses amidase (Am) activity. In general, amidases convert the amide product of Reaction 1 to the corresponding carboxylic acid plus ammonia according to Reaction 2:RCONH2RCOOH+NH3  Reaction 2
A wide variety of bacterial genera are known to possess a diverse spectrum of nitrile hydratase and amidase activities, including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium, and Micrococcus (Martinkova and Kren, Biocatalysis and Biotransformation, 20:73-93 (2002); Cowan et al., Extremophiles, 2:207-216 (1998)). For example, nitrile hydratase enzymes have been isolated from Pseudomonas chlororaphis B23 (Nishiyama et al., J. Bacteriol., 173:2465-2472 (1991)), Rhodococcus rhodochrous J1 (Kobayashi et al., Biochem. Biophys. Acta, 1129:23-33 (1991)), Brevibacterium sp. 312 (Mayaux et al., J. Bacteriol., 172:6764-6773 (1990)), Rhodococcus sp. N-774 (Ikehata et al., Eur. J. Biochem., 181:563-570 (1989)), and Pseudomonas putida 5B NRRL-18668 (Payne et al., Biochemistry, 36:5447-5454 (1997)).
Wild-type microorganisms known to possess nitrile hydratase activity have been used to convert nitrites to the corresponding amides. Nagasawa et al. (Appl. Microbiol. Biotechnol., 40:189-195 (1993)) have compared three microbial nitrile hydratase catalysts which have been used for commercial production of acrylamide from acrylonitrile; the nitrile hydratase activities of Brevibacterium R312 and Pseudomonas chlororaphis B23 were not stable above 10° C., compared to the nitrile hydratase activity of Rhodococcus rhodochrous J1. Cowan et al. (supra) reported that many mesophilic nitrile hydratases are remarkably unstable, having very short enzyme activity half-lives in the growth temperature range of 20-35° C. In addition to temperature instability, microbial catalysts containing a nitrile hydratase can be susceptible to inactivation by high concentrations of certain substrates such as acrylonitrile. In commercial use, the concentration of acrylonitrile was maintained at 1.5-2 wt % when using Brevibacterium R312 and P. chlororaphis B23 catalysts, while a concentration of up to 7 wt % was used with R. rhodochrous J1 (Nagasawa et al., supra). Similarly, Padmakumar and Oriel (Appl. Biochem. Biotechnol., 77-79:671-679 (1999)) reported that Bacillus sp. BR449 expresses a thermostable nitrile hydratase, but when used for hydration of acrylonitrile to acrylamide, inactivation of the enzyme occurred at concentration of acrylonitrile of only 2 wt %, making this catalyst unsuitable for commercial applications. Webster et al. (Biotechnology Letters, 23:95-101 (2001)) compare two Rhodococcus isolates as catalysts for ammonium acrylate production (one with only a nitrilase activity, and one with only a combination of nitrile hydratase and amidase activities), and concluded that the catalyst having a combination of nitrile hydratase and amidase activities was less preferred due to (a) difficulty in inducing the two enzymes in the required ratio, (b) the susceptibility of the two enzymes (nitrile hydratase and amidase) to deactivation by acrylonitrile, and (c) inhibition of the two enzymes by the respective products.
The hydration of aromatic and heteroaromatic nitrites to the corresponding amides has been reported using the nitrile hydratase activity of Rhodococcus rhodochrous AJ270 (A. Meth-Cohn and M. Wang, J. Chem Soc., Perkin Trans. 1, (8):1099-1104 (1997)), where significant subsequent conversion of the amide to the corresponding acid by amidase was also observed. The nitrile hydratase activity of Rhodococcus rhodochrous J1 was used to convert a variety of aromatic and heteroaromatic nitrites to the corresponding amides with 100% molar conversion (J. Mauger et al., Tetrahedron, 45:1347-1354 (1989); J. Mauger et al., J. Biotechnol., 8:87-96 (1988)); an inhibitory affect of certain nitriles on the nitrile hydratase was overcome by maintaining a low concentration of the nitrile over the course of the reaction. U.S. 20040142447 describes the use of several Rhodococcus strains for the conversion of 3-cyanopyridine to nicotinamide, where the Rhodococcus strains were relatively stable and had a relatively low Km value for 3-cyanopyridine when compared to previously-reported microbial cell catalysts.
In addition to the use of wild-type organisms, recombinant organisms containing heterologous genes for the expression of nitrile hydratase are also known for the conversion of nitrites. For example, Cerebelaud et al. (WO 9504828) teach the isolation and expression in E. coli of nitrile hydratase genes isolated from C. testosteroni. The transformed hosts effectively convert nitrites to amides, including substrates which consist of one nitrile and one carboxylate group. Endo et al. disclose the production of an E. coli transformant which expresses the nitrile hydratase of Rhodococcus N-771 (U.S. Pat. No. 6,316,242 B1). Similarly, Beppu et al., (EP 5024576) disclose plasmids carrying both nitrile hydratase and amidase genes from Rhodococcus capable of transforming E. coli where the transformed host is then able to use isobutyronitrile and isobutyramide as enzymatic substrates. A stereoselective nitrile hydratase from Pseudomonas putida 5B has been overproduced in E. coli(Wu et al., Appl. Microbiol. Biotechnol., 48:704-708 (1997); U.S. Pat. No. 5,811,286).
Genes encoding enzymes having amidase activity have also been cloned, sequenced, and expressed in recombinant organisms. For example, Azza et al., (FEMS Microbiol. Lett., 122:129 (1994)) disclose the cloning and over-expression in E. coli of an amidase gene from Brevibacterium sp. R312 under the control of the native promoter. Similarly, Kobayashi et al., (Eur. J. Biochem., 217:327 (1993)) teach the cloning of both a nitrile hydratase and amidase gene from R. rhodococcus J1 and their co-expression in E. coli. Wu et al. (DNA Cell Biol., 17:915-920 (1998); U.S. Pat. No. 6,251,650) report the cloning and overexpressing of a gene for amidase from Pseudomonas putida 5B in E. coli. 
Applicants have previously isolated Comamonas testosteroni 5-MGAM-4D (ATCC 55744; U.S. Pat. No. 5,858,736 and U.S. Pat. No. 5,922,589). Comamonas testosteroni 5-MGAM-4D has been shown to contain thermally-stable, regiospecific nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities useful in the conversion of a variety of nitriles to their corresponding amides and carboxylic acids. Methods illustrating the utility of the Comamonas testosteroni 5-MGAM-4D nitrile hydratase and amidase activities have been described previously by the Applicants. These uses include regio-selective preparation of lactams from aliphatic α,ω-dinitriles (U.S. Pat. No. 5,858,736), bioconversion of 3-hydroxynitriles to 3-hydroxyacids (U.S. 2002/0039770 A1), and bioconversion of methacrylonitrile and acrylonitrile to their corresponding carboxylic acids (U.S. Ser. No. 10/067,652), hereby incorporated by reference. However, the isolation and recombinant expression of the nucleic acid fragments encoding the nitrile hydratase and amidase from Comamonas testosteroni 5-MGAM-4D has been elusive.
The problem to be solved is to provide the genes and encoding for the thermally-stable, regio-selective nitrile hydratase and amidase enzymes from Comamonas testosteroni 5-MGAM-4D and to provide transformants expressing these catalysts.
Additionally, the development of industrial processes which employ microbial catalysts having nitrile hydratase/amidase activities to efficiently manufacture amides or carboxylic acids has proved difficult. Many methods using enzyme catalysts to prepare these products from the corresponding nitrites do not produce and accumulate the product at a sufficiently high concentration to meet commercial needs, or are subject to enzyme inactivation (requiring a low concentration of nitrile over the course of the reaction) or product inhibition during the course of the reaction.
The additional problem to be solved continues to be the lack of facile microbial catalysts to convert nitrites to the corresponding amides or acids in a process characterized by high yield, high concentration, and high selectivity, and with the added advantages of low temperature and energy requirements and low waste production when compared to known chemical methods of nitrile hydrolysis. Comamonas testosteroni 5-MGAM-4D expresses a thermally-stable, regio-selective nitrile hydratase as well as a thermally-stable amidase. An enzyme catalyst having only the nitrile hydratase activity of Comamonas testosteroni 5-MGAM-4D would be highly useful in applications where only the amide product from nitrile hydration is desired.